The goal of therapeutic gene delivery is significantly closer due to scientific progress made in the last 10 years. One of the most notable successes of the gene therapy effort is the treatment of several children with X-linked severe combined immunodeficiency syndrome (X-SCID). However, the therapy required ex-vivo retroviral transduction, and in vivo selection of transduced cells making the approach limited to the treatment of a small number of diseases. The development of Adeno-Associated Virus (AAV) vectors for gene delivery is another significant advance and has resulted in substantial increases in the efficiency of gene delivery, making in vivo gene therapy a possibility for a variety of genetic diseases. Because AAV vectors hold such promise for therapeutic gene delivery, we must begin to lay the groundwork for safety studies that might influence vector design and the selection of tissue targets. Since both integration frequency and integration- site distribution were important aspects of the development of leukemia in some X-SCID gene therapy patients, these are obvious areas of focus for assessing vector safety. AAV vectors are different than retroviruses because they depend entirely on host cell proteins for vector genome-processing, transcription and integration. This dependence raises the question of whether AAV vectors influence normal cell functions during transduction. AAV vectors clearly influence DSB repair pathways in the cell as evidenced by the fact that they are incorporated at repair sites. An important remaining question is whether AAV vectors alter cellular repair pathways even when they are not incorporated in the reaction. Understanding which repair pathways are affected, and the extent that AAV vectors influence DNA repair is essential for the appropriate design and analysis of safety studies. Experiments described here focus first on integration frequencies in different mouse tissues, and second on the affect of AAV vectors on DNA repair pathways in the cell. Finally, we propose to alter integration site location by generating repair substrates at benign genomic locations that are less mutagenic than DNA DSBs. <Relevance to public health>This research focuses on safety issues related to the administration of viral vectors for therapeutic gene delivery and will help advance the treatment of genetic disease.